Proteome research is underway to examine the states of diseases and to explore the causes of the diseases through extensive examination of in vivo proteins. For example, research for determination of proteins that can serve as disease markers, such as cancer marker proteins, is underway.
Furthermore, in recent years, transcriptome analysis has been actively conducted for extensive analysis of RNA expression, as a result of improved DNA chip technology. However, RNA expression profiles do not always agree with the protein expression profiles. The correlation between the two is said to be 50% or less. To achieve the aforementioned purpose, extensive analysis of both protein(s) and RNA is also important. Attempts to elucidate protein functions have been made using a two-dimensional electrophoresis method that has currently been significantly developed, mass spectrometry, and high throughput analysis equivalent to genome analysis such as protein chip analysis.
As described above, methods for analyzing proteins more efficiently are required with the advancement of protein research. Establishment of technology for conveniently and rapidly analyzing a great variety of proteins is important.
Electrophoresis has been broadly employed as a method for analyzing proteins. In proteome research, such electrophoresis is used for comparing protein components contained in normal tissues with those contained in disease tissues (differential display) or for preparing proteins to be used for protein identification, for example.
Electrophoresis requires staining (labeling) proteins for detection. Staining methods are largely classified into two methods.
One method is a pre-staining method that involves labeling a protein before electrophoresis. The other method is a post-staining method that involves labeling a protein after electrophoresis. An example of the former method is an Ettan DIGE method. Examples of the latter method include a Bio-Safe CBB staining method and a Sypro Ruby staining method. The characteristics of these methods are each listed in the following Table 1.
TABLE 1OperationalTotalBindingPre-stainingstepsoperationStainingmode withor(time required:timeDetectionmethodproteinpost-stainingminute)(minute)methodDefectsEttanCovalentPre-stainingReaction 40 + αFluorescenceAfterDIGEbinding(30) →electrophoresis,stop (10)analysis using amassspectrometercannot beperformed.Bio-SafeNon-covalentPost-stainingImmobilization120 + αVisiblePoor detectionCBBbinding(30) →sensitivity(binding notstaining (60)mediated by→SDS)washing (30)SyproNon-covalentPost-stainingImmobilization240 + αFluorescenceGood detectionRubybinding(30) →sensitivity.(binding notstaining (180)However,mediated by→staining andSDS)washing (30)washingprocessesrequire longtime (half a dayto one day insome cases).
In the case of the Ettan DIGE method, proteins in a sample are labeled in advance with a fluorescent reagent via covalent binding before electrophoresis. This method requires a short total operation time and has high general versatility, but it is problematic in that a plurality of fluorescent reagents bind to one protein molecule and the number of such fluorescent reagent bound to each protein differs depending on the protein in question. Hence, proteins separated by electrophoresis cannot be subjected to mass spectrometry in proteomic analysis.
In the cases of the Bio-Safe CBB method and the Sypro Ruby method, the post-staining methods, a series of complicated steps such as:    a. immobilization of proteins on gel and removal of SDS;    b. staining of proteins with a fluorescent reagent;    c. washing off of excess fluorescent reagent; and    d. detectionmust be performed after completion of electrophoresis. In particular, when steps “a” and “c” are omitted or the operation time is shortened, problems arise, such as proteins being insufficiently stained with a fluorescent reagent and the background fluorescence intensity being increased so as to make it difficult to detect protein spots.
The present inventors have reported a reagent capable of forming a complex with a protein to vary the resulting fluorescence or luminescence, so as to enable rapid and convenient analysis of the protein (JP Patent Application No. 2005-167613).